Abstract

The development of techniques allowing the growth of primordial follicles to mature follicles in vitro has much potential for both reproductive medicine and developmental research. However, human primordial and preantral follicles fail to grow after isolation from the surrounding ovarian stroma. Granulosa cells (GCs), which normally undergo apoptosis after ovulation, contain a subpopulation of ovarian follicular cells remaining viable in vitro over prolonged time periods when cultured in the presence of leukaemia inhibiting factor (LIF). However, when cultured as monolayers, they progressively lose all their characteristics, such as follicle-stimulating hormone receptor (FSHR) and cytochrome P450-aromatase. Here, we describe a three-dimensional (3D) culture system containing type I collagen, which, together with LIF, allowed the survival and growth of a subpopulation of GCs isolated from mature ovarian follicles and supported them to proliferate into spherical structures exhibiting steroidogenic capacity, as demonstrated by P450-aromatase and 3-hydroxysteroid dehydrogenase. After transplantation into the ovaries of immuno-deficient mice, these cells became localized preferentially within antral follicles and the prolonged expression of FSHR was confirmed as well. With this optimization of the culture conditions an environment was created, which acts as a niche closely mimicking the development of early ovarian follicles in vitro.